Cytotoxic protein hybrid and process for the preparation thereof

ABSTRACT

A cytotoxic protein hybrid obtained by covalently bonding an immunoglobulin or its fragment, which is capable of linking selectively with an antigen possessed by a cell to be destroyed, to a protein, which is obtained from Phytolacca americana and has an activity to terminate protein synthesis. This protein hybrid displays remarkable cytotoxicity against target cells.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel cytotoxic protein hybrid and aprocess for the preparation thereof. More particularly, this inventionrelates to a novel cytotoxic protein hybrid which has a moietyconsisting of an immunoglobulin capable of binding selectively to aspecific antigen possessed by a cell to be killed (hereinafter referredto as a target cell) or consisting of its fragment having a portionwhich binds to such antigen and a moiety consisting of a protein(hereinafter referred to as PAP) which is a purified extract ofPhytolacca americana capable of inhibiting protein synthesis and aprocess for the preparation of the same.

2. Description of the Prior Art

Attempts and trials have heretofore been made to bond an immunoglobulin,which is capable of binding selectively to a target cell, with a varietyof cytotoxic substances with the purpose of destroying certain kinds ofcells selectively. As cytotoxic substances to be bonded to theimmunoglobulin, antitumor drugs, enzyms, or toxins have so far been used(Refer to T. Ghose et al., J. Natl. Cancer Inst., Vol. 61, pp. 657-676,1978). However, since these substances essentially have nonselectivecytotoxicity, sufficient selective toxicity is not expected of them whenthey are coupled to a selective immunoglobuln. The inventors of thepresent invention have recently succeeded in obtaining a protein hybridhaving outstanding selective toxicity by first separating fragment A,which has a lethal activity to inhibit protein synthesis, and fragmentB, which binds to a variety of cells nonselectively, from diphtheriatoxin and then by cross-linking said fragment A and fragment Fab' ofimmunoglobulin (Refer to Y. Masuho et al., Biochem. Biophys. Res.Commun., Vol. 90, pp. 320-360, 1979 and Japanese Patent ApplicationLaid-open No. 136,235/80). It is known that, besides diphtheria toxin,fragments having an activity to inhibit protein synthesis can beobtained from plant toxin ricin, abrin, modeccin, etc. (refer toJapanese Patent Application Laid-open No. 49,321/80, for instance). Ahybrid of a fragment of such toxin and immunoglobulin or its fragmenthas a few demerits since it uses protein arising from toxin. The firstof such demerits is that it is very difficult to isolate fragmentshaving an activity to inhibit protein synthesis and purify them. Thesecond is a problem, which somewhat relates to the first one, thatintact toxin is apt to contaminate the fragment thus, because of theextremely potent cytotoxicity of the toxin, making the cytotoxicity ofthe hybrid prepared from such fragment nonselective, namely the hybriddestroys not only the target cells but also other, non-target cellsnonselectively, even if the amount of toxin coming into the fragment isvery small.

SUMMARY OF THE INVENTION

The present inventors have found that PAP of Phytolacca americana, asingle polypeptide chain, which by itself can not enter into cells andaccordingly its cytotoxicity is very low, can be used without beingsubjected to any treatment, especially it is not required to befragmentized before use, and accordingly its separation and purificationare very easy. Based on the knowledge the inventors obtained asmentioned above, we devoted deep study to the preparation of a novelprotein hybrid which can selectively send PAP into a target cell andhave come to invent a protein hybrid having cytotoxicity with highselectivity free from demerits found with the prior arts to work out thepresent invention.

This invention is directed to a cytotoxic protein hybrid comprising animmunoglobulin or its fragment which is capable of binding selectivelyto a specific antigen possessed by a cell to be destroyed, covalentlybonded to PAP, which is obtained from Phytolacca americana and has anactivity to terminate protein synthesis, and also to a process for thepreparation of a cytotoxic protein hybrid comprising bonding animmunoglobulin, or its fragment, to PAP with the use of a cross-linkingagent.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a protein elution pattern obtained by Sephadex G 150(superfine) column chromatography conducted for the reaction mixtureprepared in Example 1, (f),

FIG. 2 shows SDS-PAGE patterns obtained with a 6% gel. Disc 1, Fab';disc 2, PAP; disc 3, peak I of FIG. 1; disc 4, peak II of FIG. 1; disc5, peak III of FIG. 1; disc 6, peak IV of FIG. 1; disc 7, a reductionproduct of peak I of FIG. 1; and disc 8, a reduction product of peak IIof FIG. 1. Portions shaded with oblique lines are bands of lowconcentration.

FIG. 3 presents the result of the investigation made on the cytotoxicityof the protein hybrid against L 1210 cells in Example 1, (g), and thegraph shows the number of the viable cells after the 42-h incurbationversus concentration of the protein hybrid or its constituent proteinadded thereto.

FIG. 4 shows SDS-PAGE patterns obtained with a 6% gel. Disc 1, F(ab')₂ ;disc 2, PAP; disc 3, a hybrid of F(ab')₂ and PAP obtained in Example 4,(b); disc 4, a reduction product of the said hybrid with the use of2-mercaptoethanol. Portions shaded with oblique lines are bands of lowconcentration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present invention, what is referred to as an immunoglobulin (ahoming part of the cytotoxic protein hybrid) which is capable of bindingselectively to a specific antigen of a cell to be destroyed includes thefollowing. It is an immunoglobulin prepared from antisera obtained fromanimals such as man, monkey, horse, cow, goat, sheep, rabbit, guineapig, hamster, rat, mouse, etc. which are immunized with such targetcells as tumor cells or certain lymphocytes or tissues which contain anyof them by such a publicly known method as ethanol fractionation,ammonium sulfate fractionation, ion exchange or molecular sieve columnchromatography. It is also a monoclonal antibody obtained fromantibody-producing lymphocytes transformed by a carcinogen such ascarcinogenic virus or from hybridomas which are prepared by allowingantibody-producing lymophocytes obtained from an animal immunized withthe target cells to fuse with myelona cells. An immunoglobulin, which isobtained by cleaving its binding to the target cell by use of a surfaceactive agent, etc. and is specific to said target cell, is also includedin the immunoglobulins of the present invention.

It is known that the immunoglobulin falls under five classes, i.e. IgG,IgA, IgM, IgD, and IgE, and each class consists of several subclasses.But they are identical in their basic structure in that it consists oftwo heavy chains and two light chains and that they are composed of aFab moiety which has an activity of binding to an antigen and an Fcmoiety which has an effector activity. However, IgM exists as a pentamerand IgA partially as a dimer and it is desirable from the viewpoint oftissue permeability of the cytotoxic protein hybrid to reduce them tomonomers with mercaptan prior to use as a homing part of the hybrid.

As a homing part of the cytotoxic protein hybrid, the whole of theimmunoglobulin may be used, but it is preferable to use a fragment whichhas an antigen-binding part but not an Fc part. This is because in thehybrid, which contains an Fc part in it, the Fc part encouragesnonspecific adsorption on cells other than the target cells and thebinding to an Fc receptor on the cell membrane, thus reducing thefunction of the cytotoxic protein hybrid to select cells to be killed.Furthermore, since the antigenicity of the immunoglobulin as axenogeneic protein is especially strong at its Fc part, a fragment ofthe immunoglobulin having no Fc part is preferable to be used as ahoming part of the cytotoxic protein hybrid from viewpoint of loweringthe antigenicity of the protein hybrid. The digestion of animmunoglobulin with a proteolytic enzyme such as papain, trypsin,chymotrypsin, plasmin, etc. generally gives what is called Fab fragmenthaving one variable region. Also the digestion of an immunoglobulin withpepsin or trypsin, depending upon the conditions, gives what is calledF(ab')₂ fragment having two variable regions. This fragment furtherturns to a monovalent Fab' fragment when it is treated with mercaptan.When the immunoglobulin is decomposed while being denatured, it gives avariable region only. These fragments arising from immunoglobulins canall be used as a homing part of the protein hybrid of the presentinvention disregard of the class and subclass to which the materialimmunoglobulins belong.

What is called PAP, a protein capable of inhibiting protein synthesisobtained from Phytolacca americana, in the present invention is aprotein, which has the molecular weight of about 27,000 and comprisessingle polypeptide chain, can be extracted from Phytolacca americana andpurified according to a publicly known method, such as the methodproposed by J. D. Irvin (Archives of Biochemistry and Biophysics, Vol.169, pp. 522-528, 1975). It has a strong activity to inhibit thesynthesis of polyphenylalamine with the use of ribosome of reticulocyte.PAP can be extracted not only from the leaves of Phytolacca americanabut also from its seeds and roots.

The cytotoxic protein hybrid of the present invention is prepared bycross-linking an immunoglobulin or its fragment and PAP of Phytolaccaamericana covalently. This kind of covalent cross-linking may beeffected by directly binding both constituent elements by amide bondwith the use of a carbodiimide reagent such as dicyclohexylcarbodiimide(DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDCI), etc. as a condensing agent. Also both constituent elements maybe linked with the use of a cross-linking reagent having a plural numberof the same cross-linking functional groups in the molecule.Glutaraldehyde, toluenediisocyanate,2,2'-dicarboxy-4,4'-azeophenylenediisocyanate, diethylmalonimidatehydrochloride, for instance, may be mentioned as a cross-linking agentfor such a use. A hybrid can be prepared by covalently binding animmunoglobulin or its fragment and PAP according to the methodsmentioned above; however, it is difficult to obtain a desired hybrid,because there also occur intramolecular cross-linking, cross-linkingbetween immunoglobulins or their fragments, or cross-linking betweenPAP's. Therefore, a preferable method is one in which firstly one of theconstituent elements, i.e. an immunoglobulin or its fragment and PAP, ismade to react with a cross-linking agent with the use of a cross-linkingreagent which has a several number (preferably one each) of two kinds ofcross-linking functional groups different from each other in themolecule, and then the reaction product thus obtained is made to reactwith the other of the constituent elements. In this case, theabovementioned reaction can be carried out after cross-linkingfunctional groups are introduced to both or either of the constituentelements. Under the method mentioned above, the cytotoxic protein hybridexpressed by formula (I) or (II) can be obtained:

    Ab--(X.sub.1).sub.t --PAP).sub.n                           (I)

    (Ab--(X.sub.1).sub.t --PAP                                 (II)

(where Ab expresses an immunoglobulin or its fragment, PAP indicates aprotein having an activity to inhibit protein synthesis obtained fromPhytolacca americana; X₁ is a divalent organic groups; t stands for 0 or1; and n indicates an integer of 1 to 3).

X₁ is a divalent organic group arising from a cross-linking agent;however, of the cytotoxic protein hybrids, those hybrids expressed bythe following formulas (III), (IV), (V), and (VI) are especiallypreferable from the viewpoint of preparation, separation, purification,and activity: ##STR1## (where definitions of Ab, PAP, and n are the sameas those given in cases of formulas (I) and (II); X₂, X₃, X₄, and X₅indicate divalent organic groups; S₁ and S₂ stand for sulfur atoms; pand q are the same or different from each other, indicating 0 or 1).

In the abovementioned formulas (III) to (VI), when p=0, S₁ is a sulfuratom arising from an immunoglobulin or its fragment and when p=1, S₁ isa sulfur atom introduced by a cross-linking agent. S₂ is a sulfur atomintroduced by a cross-linking agent. In formulas (III) and (IV), whenq=0, sulfur atoms S₁ and S₂ link together directly to form a disulfidegroup. On the other hand, when q=1, sulfur atoms S₁ and S₂ link togetherthrough the medium of X₃, a divalent organic group. X₃ is a divalentorganic group arising from a cross-linking agent having two functionalgroups which react with a thiol group, for instance, a cross-linkingagent expressed by formula (VII): ##STR2## (where X₆ is a divalentorganic group) or benzoquinone. X₂ in formulas (III) to (VK) and X₄ informulas (III) and (IV) are the same or different from each other andare divalent organic groups arising;

from a cross linking agent expressed by the following formula (VIII):##STR3## (where Y indicates a monovalent organic group which can form anactive disulfide group together with a sulfur atom S linked to it; X₇ isa divalent organic group; and Z indicates an alcohol residue of activeester),

from a cross-linking agent expressed by the following formula (IX):##STR4## (where definitions of Y and X₇ are the same as those given incase of formula (VIII); Q indicates an alcohol residue of imido ester;and R represents a halogen atom),

from a cross-linking agent expressed by the following formula (X):##STR5## (where definitions of X₇ and Z are as same as those given incase of formula (VIII) and a definition of R is as same as that given incase of formula (IX)),

from a cross-linking agent (2-iminothiolactone) expressed by thefollowing formula (XI): ##STR6## from a cross-linking agent(N-acetylhomocysteine) expressed by the following formula (XII):##STR7## from a cross-linking agent (S-acetylmercaptosuccinic acidanhydride) expressed by the following formula (XIII): ##STR8## and froma cross-linking agent expressed by the following formula (XIV): ##STR9##(where definitions of X₇ and Z are the same as those given in case offormula (VIII)).

As concrete examples of monovalent organic groups which are able to forman active disulfide group together with a linked sulfur atom, a##STR10## etc. may be mentioned. There are no restrictions as to thekind of a divalent organic group expressed by X₆ or X₇ so far as it ischemically inactive; however, in general, it is suitably selected fromamong alkylene groups or phenylene groups, both having branchings ornot. As concrete examples of alcohol residue of active ester expressedby Z, ##STR11## etc. may be mentioned. As concrete examples of alcoholresidue of imido ester expressed by Q, methoxy group, ethoxy group, etc.may be mentioned. As concrete examples of halogen atom expressed by R,chlorine, bromine, etc. may be mentioned.

As concrete examples of cross-linking agent,N,N'-(1,2-phenylene)dimaleimide, N,N'-(1,4-phenylene)-dimaleimide,4,4'-bis(maleoylamino)azobenzene, and bis(N-maleimidomethyl)ether may bementioned as crosslinking agent expressed by formula (VII);N-succinimidyl 3-(2-pyridyldithio)propionate, and 2,4-dinitrophenyl3-(4-pyridyldithio)propionate as cross-linking agent expressed byformula (VIII); methyl 3-(2-pyridyldithio)propionimidate hydrochlorideas cross-linking agent expressed by formula (IX); and N-succinimidyl3-bromopropionate as cross-linking agent expressed by formula (X).

Of the cytotoxic protein hybrids of the present invention, the hybridexpressed by formula (III) or (IV) can be prepared according to themethod in which the active disulfide group is introduced to either ofthe immunoglobulin or its fragment and PAP which form the protein hybrid(hereinafter the optional one protein shall be referred to as Pro 1 andthe other protein Pro 2) and the thiol group is introduced to or formedin the other and then both proteins are cross-linked by means ofdisulfide bond or by use of a cross-linking agent. More particularly,Pro 1 is made to react with a cross-linking agent expressed by formula(VIII) or formula (IX) (reactions (1) and (2)) ##STR12## or is made toreact with a cross-linking agent expressed by formula (X), followed bythe thiosulfate ion treatment of the reaction product (XVII) (reaction3), ##STR13## or Pro 1 is made to react with a cross-linking agentexpressed by formula (XI) or (XII) to form a protein expressed byformula (XVIII) or (XIX), or with a cross-linking agent expressed byformula (XIII) followed by deacetylation of the resulting protein togive a protein expressed by formula (XX), and thus obtained protein isthen treated with a reagent which converts a thiol group into an activedisulfide group, such as ##STR14## (where a definition of Y is the sameas that in case of formula (VIII), ##STR15## to obtain Pro 1 (a proteinexpressed by formula (XV), (XVI), (XV-1), (XVI-1), (XV-2) or (XV-3), towhich an active disulfide group is introduced). On the other hand, adisulfide group of protein expressed by the following formula (XXI),(XXIII), or (XXI-1), which is prepared from the other protein Pro 2according to the abovementioned formula (1), (2), or (3), is reducedwith such a thiol reagent as 2-mercaptoethanol, dithiothreitol, etc.,(reaction (7), (8), or (9)) ##STR16## or is subjected to the first stepof the reaction expressed by formula (4) or (5), or to the first and thesecond steps of the reaction expressed by formula (6) to obtain Pro 2 towhich a thiol group is introduced (a protein expressed by formula XXII),(XXIV), (XXIV-1), or (XXI-1). ##STR17## (In the foregoing, X₇ in formula(XV), (XVI), or (XV-1) and X₇ in formula (XXII) or (XXIV) may be thesame or different from each other.) The cytotoxic protein hybrid of thepresent invention expressed by formula (III) or (IV) (in both cases q=0)can be prepared by making Pro 2, which is obtained by the introductionof a thiol group as mentioned above, react with Pro 1 which is likewiseobtained by the introduction of an active disulfide group as mentionedabove.

A protein hybrid of the present invention expressed by formula (V) or(VI) can be prepared by first making PAP react with a cross-linkingagent expressed by formula (XIV), for instance, to obtain a proteinexpressed by formula (XXV) to which a maleimide group is introduced,##STR18## and then making thus obtained protein react with animmunoglobulin or its fragment prepared according to the reactionformula (7), (8), (9), the first step of the reaction formula (4) or(5), or the first and the second steps of the reaction formula (6) towhich a thiol group is introduced.

One of the precursors of the protein hybrid of the present inventionexpressed by formula (III), (IV), (V), or (VI) is a protein having athiol group. As mentioned concretely with the chemical formulas, thethiol groups as these may include, in addition to a thiol groupintroduced from outside, a thiol group of the protein itself when theprotein has one or a thiol group obtained by reducing the disulfide bondof the protein arising from a cystine group.

In the aforementioned cross-linking reaction between an immunoglobulinor its fragment and PAP, it is desirable to use 1 to 100 moles of across-linking agent per 1 mole of protein in case an immunoglobulin orits fragment is made to react with a cross-linking agent or in case PAPis made to react with a cross-linking agent. The reaction is conductedby adding an aqueous solution of cross-linking agent or a solutionprepared by dissolving a cross-linking agent in a small amount oforganic solvent such as N,N-dimethylformamide, dimethylsulfoxide,1,2-dimethoxyethane, methanol, ethanol, acetone, etc. when thecross-linking agent is insoluble in water, to a solution prepared bydissolving an immunoglobulin or its fragment, or PAP in such a way thatthe concentration of protein in the buffer with pH adjusted to 4-9 maybe in the range of 0.5-100 mg/ml (more preferably in the range of 1-20mg/ml) at 0° to 50° C. with stirring. The reaction time varies dependingupon the reaction scale and reaction conditions and, in general, thereaction is carried out within 2 days. After the reaction is over, thecross-linking agent left unreacted is removed by dialysis or molecularsieve column chromatography to obtain a solution of protein to which thecross-linking agent is introduced. With the protein thus prepared ismade to react at 0° to 50° C. a solution prepared by dissolving aprotein, which is another constituent element of the hybrid (or aprotein to which a cross-linking functional group is introduced by across-linking agent) in a buffer with the pH value of 4-9 (thepreferable range of protein concentration is the same as those mentionedabove). The separation of the hybrid from the reaction mixture and itspurification can be carried out following the ordinary procedure such asmolecular sieve column chromatography. The hybrid can also be preparedby adding a solution of a protein, which makes one of the constituentelements of the hybrid and to which a cross-linking agent is introduced,to a solution of a protein which makes another of the constituentelements of the hybrid. Furthermore, the reaction conditions to beadopted in the following cases are the same as the reaction conditionsadopted in the abovementioned case; a case where a cross-linking agentis made to react with a protein; in case where a protein, to which across-linking agent is introduced, is treated with a low molecularweight reagent to be converted to a protein having a specificcross-linking functional group (for instance, a case where an activedisulfide group introduced by a cross-linking agent is converted into athiol group), or a case where an immunoglobulin or its fragment or PAPis directly converted into an activated derivative with the use of a lowmolecular weight reagent (for instance, a case where a thiol group offragment Fab' of the immunoglobulin is converted into an activedisulfide).

The present invention is described in detail by the following examples.

EXAMPLE 1 (a) Extraction of protein (PAP) having activity to inhibitprotein synthesis from leaves of Phytolacca americana and purificationthereof

A homogenate was obtained by homogenizing a mixture of 500 g ofPhytolacca americana leaves and 500 ml of water. The homogenate wassubjected to ammonium sulfate fractionation, chromatography on DEAEcellulose, and chromatography on phosphocellulose according to themethod of Irvin (Archives of Biochemistry and Biophysics, vol. 169, pp.522-528, 1975) to obtain purified PAP. Thus purified preparation gave asingle band at the site of molecular weight of about 27,000 on sodiumlauryl sulfate-polyacrylamide gel electrophoresis (hereinafter referredto as SDS-PAGE) as shown by disc 2 in FIG. 2. SDS-PAGE was conductedaccording to the method of K. Weber and M. Osborn (J. Biol. Chem., Vol.244, pp. 4406-4112, 1969).

It was confirmed by SDS-PAGE that PAP could also be obtained from theseeds and roots of Phytolacca americana by extraction and purification.In the following experiments, the purified preparation obtained from itsleaves was used.

(b) Preparation of N-[3-(2-pyridyl)dithiopropionyl]-PAP

0.09 ml of ethanol solution containing 9 m M N-succinimidyl3-(2-pyridylthio)propionate (SPDP) was added to 1.6 ml of 0.02 Mphosphate buffer--0.14 M sodium chloride--1 m Methylenediaminetetra-acetic acid (hereinafter referred to as EDTA)solution (pH 7.5) containing 8.1 mg of PAP extracted and purifiedaccording to the preceding (a), and the mixture was made to react atroom temperature for 30 minutes. The reaction product was put toSephadex G25 column chromatography on said phosphate buffer (pH 7.5) toremove the excess reagent. PAP, into which about 2N-[3-(2-pyridyl)dithiopropionyl] groups on the average were introduced,was thus obtained.

(c) Preparation of immunoglobulin having specificity to mouse leukemia L1210

Mouse leukemia L 1210 cells transplanted successively on DBA/2Cr micewere taken out of the ascites of a DBA/2Cr mouse. An emulsion preparedfrom about 10⁸ of those cells and Freund's complete adjuvant wasintravenously injected into rabbits. After that 10⁶ L 1210 cells,together with the adjuvant, were subcutaneously injected three times atintervals of one week, and the rabbits were bled seven days and ten daysafter the day of final injection. The bloods thus obtained were mixed,and serum was separated and heated at 56° C. for 30 minutes to beinactivated. 200 ml of a saturated aqueous solution of ammonium sulfatewas added to 200 ml of thus obtained anti-L 1210 antiserum, and theresulting precipitate was separated by means of centrifugation. Theprecipitate thus separated was dissolved in 50 ml of 0.01 M phosphatebuffer (pH 7.6) and further dialyzed against the same buffer. Thedialyzate was put to DEAE cellulose column chromatography (column size 3cm×94 cm) equilibrated with the same buffer to obtain a solutioncontaining anti-L 1210 IgG as an unadsorbed fraction.

(d) Separation of fragment F(ab')₂ from immunoglobulin

1.2 g of anti-L 1210 IgG obtained in the preceding (c) was dissolved in40 ml of 0.1 M acetate buffer (pH 4.5), to which 24 mg of pepsin wasadded to effect peptic digestion at 37° C. for about 18 hours. Thedigestion product was put to Sephadex G200 column chromatography (columnsize 3.5 cm×140 cm) over saline to take out a protein eluted atmolecular weight of about 100,000. It was confirmed that this was purefragment F(ab')₂ by SDS-PAGE as shown in FIG. 4, disc 1.

(e) Preparation of fragment Fab'

0.02 ml of 150 m M aqueous 2-mercaptoethanol solution was added to 2.0ml of 0.01 M tris.hydrochloride--0.14 M sodium chloride--2 m M EDTAsolution (pH 8.3) containing 18.4 mg of fragment F(ab')₂ obtained in thepreceding (d), and the mixture was subjected to the reduction at 37° C.for 1 hour. After the reaction was over, the solution was put toSephadex G25 column chromatography (column size 1.0 cm×20 cm)equilibrated with 5 m M acetate buffer--0.14 M sodium chloride--1 m MEDTA solution (pH 5.5) (hereinafter referred to as ANE buffer) to remove2-mercaptoethanol, thus giving fragment Fab' having 1 thiol group (Referto FIG. 2, disc 1).

(f) Preparation of hybrid of fragment Fab' and PAP covalently bonded bybonds containing disulfide bond

2.0 ml of 0.02 M phosphate buffer--0.14 M sodium chloride--1 m M EDTAsolution (pH 7.5) containing 6.1 mg ofN-[3-(2-pyridyl)dithiopropionyl]-PAP was mixed with 1.0 ml of theabovementioned phosphate buffer containing 4.1 mg of fragment Fab'having 1 thiol group and the mixture was allowed to react at roomtemperature for 18 hours. The reaction solution was chromatographed onSephadex G 150 (superfine) (1.3 cm×95 cm) in physiological saline, asthe result of which 5 peaks were found when the absorbance of thefractions was measured at 280 mm as shown in FIG. 1. The result obtainedby the analysis of these peaks on SDS-PAGE is shown in FIG. 2. In FIG. 2disc 1 shows the band of Fab', and disc 2 shows that of PAP. The maincomponent of peak I in FIG. 1 had, as shown by disc 3, molecular weightof about 120,000 and was separated into fragment Fab' (corresponding todisc 1) and PAP (corresponding to disc 2) when reduced with2-mercaptoethanol. The ratio between them is 2:1. Therefore, the proteinof peak I is a hybrid consisting of two molecules of fragment Fab' andone molecule of PAP bonded together. The protein of peak II had, asshown by disc 4, molecular weight of about 70,000 and was separated intofragment Fab' and PAP when reduced with 2-mercaptoethanol at the ratioof 1:1 as shown by disc 8. Therefore, the protein of peak II is a hybridconsisting of one molecule of fragment Fab' and one molecule of PAPbonded together. The proteins of peaks III and IV are fragment Fab' andPAP respectively both remaining not reacted as shown by discs 5 and 6.

(g) Cytotoxicity of hybrid against L 1210 cells

The cytotoxicity of the protein hydrid against the target L 1210 cellsobtained in the preceding (f) was examined.

0.10 ml of RPMI 1640 culture medium (containing 10% fetal calf serum,0.02 m M 2-mercaptoethanol, and 0.1 mg/ml of kanamycin) containing 3×10⁴/ml of L 1210 cells was placed in a 96-hole microplate, to which 0.01 mlof subject samples diluted to varied concentrations was added. Theculture was carried out at 37° C. in an atmosphere of 5% CO₂ for 42hours and then the viable cells were counted by Trypan Blue dyeexclusion method. The result: as shown by FIG. 3, Fab', PAP, and a 1:1mixture of them scarcely showed cytotoxicity even at a proteinconcentration of 10⁻⁶ M. However, the protein hybrid (the protein ofpeak II of FIG. 1) prepared in the preceding (f) showed remarkablecytotoxicity. Incidentally, another experiment showed that the proteinhybrid involved in peak I of FIG. 1 had cytotoxicity equal to orsomewhat lower than that of the protein hybrid involved in peak II.

EXAMPLE 2 (a) Preparation of PAP having thiol group introduced

2-Mercaptoethanol was added to 2.0 ml of 0.1 M tris.hydrochloride--2 m MEDTA (pH 8.3) buffer solution containing 4.8 mg ofN-[3-(2-pyridyl)-dithiopropionyl]-PAP in such an amount as to obtain afinal concentration of 5 m M. After having been reduced at 37° C. for 1hour, the reaction solution was put to Sephadex G 25 columnchromatography equilibrated with an ANE buffer to remove2-mercaptoethanol and low molecular products, thus giving PAP havingabout 2 thiol groups on the average.

(b) Preparation of Fab' having maleimide group introduced

A mixture consisting of 1.75 ml of ANE buffer containing 8.3 mg offragment Fab' prepared in Example 1, (e) and 1.75 ml of ANE buffersaturated with O-phenylenedimaleimide was allowed to react at roomtemperature for 30 minutes. Thereafter, the reaction solution was put toSephadex G25 column chromatography equilibrated with an ANE buffer toremove the reagent remaining unreacted, thus giving fragment Fab' having1 maleimide group.

(c) Preparation of hybrid by cross-linking PAP having thiol groups withfragment Fab' having maleimide group

A mixture comprising 1.9 ml of ANE buffer containing 2.7 mg of PAPhaving thiol groups, 1.8 ml of ANE buffer containing 5.2 mg of fragmentFab' having a maleimide group and 0.4 ml of 0.3 M phosphate buffer-10 mM EDTA (pH 6.5) was made to react at 4° C. for 22 hours. The reactionsolution was put to the same Sephadex G 150 (superfine) columnchromatography as in Example 1, (f), to isolate a hybrid in which Fab'and PAP were linked together at the ratio of 2:1 and a hybrid in whichFab' and PAP were linked together at the ratio of 1:1. Their molecularweight was confirmed by means of SDS-PAGE as in Example 1, (f), andtheir cytotoxicity against L 1210 cells was confirmed according to themethod of Example 1, (g).

EXAMPLE 3 (a) Preparation of PAP having maleimide group introduced

0.03 ml of N,N-dimethylformamide with 7.0 mg/ml metamaleimidobenzoicacid N-hydroxysuccinimide ester dissolved therein was added to 1.0 ml of0.1 M phosphate buffer (pH 7.0) containing 3.3 mg of PAP purifiedaccording to Example 1, (a) and the mixture was made to react at roomtemperature for 30 minutes. The reaction solution was then put toSephadex G25 column chromatography equilibrated with ANE buffer toremove the unreacted reactant, thus obtaining PAP with maleimide groupintroduced.

(b) Preparation of hybrid by cross-linking PAP having maleimide group(s)with fragment Fab' having thiol group

2.2 ml of ANE buffer containing 2.6 mg of PAP having maleimide group(s)obtained in the preceding (a) and 1.7 ml of ANE buffer containing 4.1 mgof fragment Fab' having a thiol group obtained in Example 1, (e), weremixed and made to react at room temperature overnight. This reactionsolution was put to Sephadex G 150 (superfine) column chromatography toisolate a hybrid in which 2 molecules of Fab' and 1 molecule of PAP werelinked together and a hybrid in which 1 molecule of Fab' and 1 moleculeof PAP were linked together. Several other unidentified hybrids wereobserved besides these two hybrids.

EXAMPLE 4 (a) Preparation of N-[3-(2-pyridyl)dithiopropionyl]-fragmentF(ab')₂

0.05 ml of ethanol solution containing 8 m M SPDP was added to 1.7 ml of0.02 M phosphate buffer--0.14 M sodium chloride--1 m M EDTA (pH 7.5)containing 11.2 mg of fragment F(ab')₂ prepared according to Example 1,(d), and the mixture was allowed to react at room temperature for 30minutes. The reaction solution was subjected to Sephadex G25 columnchromatography equilibrated with the abovementioned phosphate buffer toremove excess reagent, thus obtainingN-[3-(2-pyridyl)dithiopropionyl]-fragment F(ab')₂.

(b) Preparation of hybrid by cross-linkingN-[3-(2-pyridyl)dithiopropionyl]-fragment F(ab')₂ with PAP having thiolgroup(s)

2.1 ml of 0.02 M phosphate buffer--0.14 M sodium chloride--1 m M EDTAsolution (pH 7.5) containing 7.8 mg ofN-[3-(2-pyridyl)dithiopropionyl]-fragment F(ab')₂ prepared according tothe method described in the preceding (a) was mixed with 1.5 ml of ANEbuffer containing 2.0 mg of PAP having thiol groups prepared accordingto Example 2, (a), and the mixture was allowed to react at roomtemperature for 20 hours. The reaction mixture was put to Sephadex G 150(superfine) column chromatography according to Example 1, (f), and thusobtained 30th, 31st, and 32nd fractions were pooled together andsubjected to SDS-PAGE to obtain the result as shown in FIG. 4. Theprotein contained in this fraction showed a pattern of bands of disc 3,from which it was observed that it contained a product of about 120,000molecular weight and a small amount of other products of largermolecular weight besides the material fragment F(ab')₂ (whichcorresponds to disc 1). The reduction of this protein conducted at 37°C. for 1 hour with 2 m M 2-mercaptoethanol brought about itsdissociation into fragment Fab' and PAP (which correspond to disc 2). Itwas made known from these results that the main product (of about120,000 molecular weight) was a hybrid comprising fragment F(ab')₂ andPAP linked together by a disulfide bond.

(c) Cytotoxicity of hybrid

The cytotoxicity of the hybrid obtained according to the preceding (b)against the target cells L 1210 was examined. The method ofdetermination was almost as same as the one which was adopted in Example1, (g); however, the cytotoxity was judged by observing the cells in themicroplate using a phase contract microscope without using Trypan Blue.When all of the globular cells were found damaged, the result isindicated by +++; when 90-50% were found damaged, the result isindicated by ++; when 50-10% were found damaged, the result is indicatedby +; and when the result was as same as that of the control, it isindicated by -. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Cytotoxicity of hybrid of                                                     F(ab').sub.2 and PAP linked with                                              disulfide bond.                                                                                Protein                                                                       concentration (μg/ml)                                     Inhibitor          20      4       0.8  0.16                                  ______________________________________                                        Mixture of F(ab').sub.2 and PAP                                                                  -       -       -    -                                     Hybrid of F(ab').sub.2 and PAP                                                                   +++     +++     ++   -                                     Hybrid of Fab' and PAP                                                                           +++     ++      +    -                                     (according to Example 1)                                                      ______________________________________                                    

As Table 1 shows clearly, the simple mixture of F(ab')₂ and PAPdisplayed no cytotoxicity at all; however, the hybrid of F(ab')₂ and PAPdisplayed a remarkable cytotoxicity. This had an activity somewhatstronger than the hybrid of Fab' and PAP obtained in Example 1.

EXAMPLE 5 (a) Fragment F(ab')₂ having maleimide group introduced

0.05 ml of N,N-dimethylformamide with 7.0 mg/ml metamaleimidobenzoicacid N-hydroxysuccinimide ester dissolved therein was added to 1.0 ml of0.1 M phosphate buffer containing 5.2 mg of fragment F(ab')₂ preparedaccording to Example 1, (d), and the mixture was made to react at roomtemperature for 30 minutes. The reaction solution was then put toSephadex G 25 column chromatography equilibrated with ANE buffer toremove the unreacted reagent, thus obtaining fragment F(ab')₂ with amaleimide group introduced.

(b) Preparation of hybrid by cross-linking F(ab')₂ having maleimidegroup with PAP having thiol group

1.8 ml of ANE buffer containing 4.2 mg of fragment F(ab')₂ having amaleimide group obtained in the preceding (a) and 1.8 ml of ANE buffercontaining 1.9 mg of PAP having a thiol group prepared according toExample 2, (a), were mixed and made to react at room temperature for 18hours. The reaction mixture was put to Sephadex G 150 (superfine) columnchromatography. The absorbance of each fraction was determined at 280 nmand two peaks were observed as in the case of Example 4, (b). When thefraction of the first peak containing a protein of greater molecularweight was subjected to SDS-PAGE, a main product of about 120,000molecular weight, or a hybrid formed by the linkage of a maleimide groupof F(ab')₂ and a thiol group of PAP, was confirmed. The cytotoxicity ofthe fractions containing this hybrid was inspected according to Example4, (c), and it was found to have the cytotoxicity against the targetcells L 1210.

What is claimed is:
 1. A cytotoxic protein hybrid prepared by covalentlybonding an immunoglobulin, or its fragment, which is capable of bondingselectively to an antigen specific to a cell to be killed, to a proteinwhich is obtained from Phytolacca americana and capable of inhibitingprotein synthesis.
 2. A cytotoxic protein hybrid according to claim 1,which is expressed by the following formula (I) or (II):

    Ab--(X.sub.1).sub.t --PAP).sub.n                           (I)

    (Ab--(X.sub.1).sub.t).sub.n PAP                            (II)

where Ab indicates an immunoglobulin or its fragment; PAP is a proteinwhich is obtained from Phytolacca americana and capable of inhibitingprotein synthesis; X₁ is a divalent organic group; t indicates 0 or 1;and n represents an integer of 1 to
 3. 3. A cytotoxic protein hybridaccording to claim 1 or 2, which is expressed by the following formula(III) or (IV):

    Ab--(X.sub.2).sub.p --S.sub.1 --(X.sub.3).sub.q --S.sub.2 --X.sub.4 --PAP).sub.n                                              (III)

    (Ab--(X.sub.2).sub.p --S.sub.1 --(X.sub.3).sub.q --S.sub.2 --X.sub.4).sub.n PAP                                                       (IV)

where Ab indicates an immunogloublin or its fragment; PAP is a proteinwhich is obtained from Phytolacca americana and capable of inhibitingprotein synthesis; n represents an integer of 1 to 3; X₂, X₃ and X₄indicate a divalent organic group respectively; S₁ and S₂ indicate asulfur atom respectively; and p and q are either the same or differentfrom each other and represent 0 or
 1. 4. A cytotoxic protein hybridaccording to claim 1 or 2, which is expressed by the following formula(V) or (VI): ##STR19## where Ab indicates an immunoglobulin or itsfragment; PAP is a protein which is obtained from Phytolacca americanaand capable of inhibiting protein synthesis; n represents an integer of1 to 3; X₂ and X₅ indicate a divalent organic group respectively; S₁indicates a sulfur atom; and p represents 0 or 1; comprising reacting adisulfide group, which is introduced to one of the protein of animmunoglobulin or its fragment and the protein which is obtained fromPhytolacca americana and is capable of inhibiting protein synthesis,with a thiol group which is either self-produced or introduced into theother of said proteins.
 5. A process for preparing a cytotoxic proteinhybrid expressed by the following formula (III-1) or (IV-1):

    Ab--(X.sub.2).sub.p --S.sub.1 --S.sub.2 --X.sub.4 --PAP).sub.n (III- 1)

    (Ab--(X.sub.2).sub.p --S.sub.1 --S.sub.2 --X.sub.4).sub.n PAP (IV-1)

where Ab indicates an immunogloublin or its fragment; PAP is a proteinwhich is obtained from Phytolacca americana and capable of inhibitingprotein synthesis; n represents an integer of 1 to 3; X₂ and X₄ indicatea divalent organic group, respectively; S₁ and S₂ indicate a sulfur atomrespectively; and p represents 0 or 1; comprising linking animmunoglobulin or its fragment, which has a thiol group eitherself-produced therein or introduced thereto, with a protein, which isobtained from Phytolacca americana, capable of inhibiting proteinsynthesis, and has a thiol group introduced thereto, by use of across-linking agent having two functional groups capable of reactingwith thiol groups.
 6. A process for preparing a cytotoxic protein hybridexpressed by the following formula (III-2) or (IV-2):

    Ab--(X.sub.2).sub.p --S.sub.1 --X.sub.3 --S.sub.2 --X.sub.4 --PAP).sub.n (III- 2)

    Ab--(X.sub.2).sub.p --S.sub.1 --X.sub.3 --S.sub.2 --X.sub.4).sub.n PAP (IV-2)

wherein Ab indicates an immunogloublin or its fragment; PAP is a proteinwhich is obtained from Phytolacca americana and capable of inhibitingprotein synthesis; n represents an interger of 1 to 3; X₂, X₃ and X₄indicate a divalent organic group respectively; S₁ and S₂ indicate asulfur atom respectively; and p is 0 or 1; comprising linking animmunoglobulin or its fragment, which has a thiol group eitherself-produced therein or introduced thereto, with a protein, which isobtained from Phytolacca americana, capable of inhibiting proteinsynthesis, and has a thiol group introduced thereto, by use of acrosslinking agent having two functional groups capable of reacting withthiol groups.
 7. A process for preparing a cytotoxic protein hybridexpressed by the following formula (V) or (VI): ##STR20## where Abindicates an immunoglobulin or its fragment; PAP is a protein which isobtained from Phytolacca americana and capable of inhibiting proteinsynthesis; X₂ and X₅ indicate a divalent group respectively; S₁indicates a sulfur atom; and p is 0 or 1; comprising reacting animmunoglobulin or its fragment having a thiol group either self-producedtherein or introduced thereto with a protein which is obtained fromPhytolacca americana, capable of inhibiting protein synthesis, and has amaleimide group introduced thereto.